Voltage-activated potassium channels serve several critical functions in all excitable cells (brain, heart, endocrine and muscle), including repolarization of action potentials and control of rhythmic firing patterns. Channel properties that control the functional outcome of channel activity include current magnitude and the rate of channel gating events (activation, inactivation and deactivation). The functionally rich outer vestibule/selectivity filter region of the potassium channel pore has been considered to have just a single conformation in the open state, and have little or no role in the modulation of open channel function. Recently, we described a novel mechanism by which current magnitude, activation rate and inactivation rate, as well as both internal and external channel pharmacology, are modulated by relevant changes in external potassium concentration. We demonstrated that changes in these channel properties, which can be substantial, result from a previously unknown type of conformational change that occurs in the outer vestibule of the pore. Furthermore, this conformational change in the outer vestibule is observed only in channels that display properties consistent with a "structurally flexible" selectivity filter region of the pore. These results suggest the possibility that, in contrast to what has been previously believed, the permeation pathway in some ion channels has a significant degree of "structural flexibility," and that this "flexibility" can markedly affect open channel function. Neither the nature of the conformational change, nor a detailed understanding of how it is regulated, can be obtained solely with electrophysiological techniques. The goal of this application is to integrate two sophisticated fluorescence techniques, fluorescence quenching and fluorescence resonance energy transfer (FRET), with patch clamp electrophysiology in our lab. This will allow us to directly examine the nature of the conformational change, and the mechanisms that control the conformational change. It will also allow us to test the fundamentally novel hypothesis that differences in "structural flexibility" of the permeation pathway underlie, in part, differences in functional regulation of closely related ion channels. The ability to incorporate this technology into our research will provide a new and enhanced approach for our study of ion channel mechanisms, and will allow us to collect preliminary data necessary for subsequent funding.